CN1306428A - Inhibitors of transcription factor NF-KB - Google Patents

Abstract

The present invention provides aminoindanone inhibitors of transcription factor NF-kb and pharmaceutically acceptable salts, hydrates, and solvates thereof, pharmaceutical compositions of these compounds, and methods of treating diseases in which NF-kb activation is implicated. More particularly, the present invention provides methods of treatment for a variety of diseases associated with NF-KB activation, including inflammatory diseases, particularly rheumatoid arthritis, inflammatory bowel disease, and asthma, skin diseases, including psoriasis and acute dermatitis, autoimmune diseases, tissue and organ rejection, Alzheimer's disease, stroke, atherosclerosis, restenosis, cancer, including Hodgkin's disease, and certain viral infections, including AIDS, osteoarthritis, osteoporosis, and Ataxia Telangiestasia, by administering a compound of the present invention to a patient in need of such treatment.

Description

Inhibitor of transcription factor NF-κB

field of invention

The present invention generally relates to methods of inhibiting the transcription factor NF-κB using aminoindanones. Such compounds are especially useful in the treatment of diseases in which NF-κB activation is involved. More specifically, these compounds inhibit IκB phosphorylation and subsequent degradation. This class of compounds is useful in the treatment of a variety of diseases associated with NF-κB activation, including inflammatory diseases; especially rheumatoid arthritis, inflammatory bowel disease and asthma; skin diseases including psoriasis and atopic dermatitis; autoimmune Disease; tissue and organ rejection; Alzheimer's disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkin's disease; and certain viral infections, including AIDS; osteoarthritis; bone loose; and Ataxia Telangiestasia.

Background of the invention

Recent advances in scientific research on mediators of acute and chronic inflammatory diseases and cancer have led to new strategies to explore effective treatments. Traditional approaches include directly targeted interventions, such as the use of specific antigens, receptor antagonists, or enzyme inhibitors. The recent breakthrough elucidation of the regulatory mechanisms of transcription and translation of various mediators has increased interest in therapeutic approaches involving the transcriptional level of genes.

NF-κB belongs to a family of substances closely related to dimeric transcription factor complexes composed of various combinations of Rel/NF-κB family polypeptides. This family of substances includes five unique gene products in mammals, RelA (p65), NF-κB1 (p50/p105), NF-κB2 (p49/p100), c-Rel, and RelB, all of which can form hybrids. Polymeric or homopolymeric dimers. These proteins share a highly homologous 300-amino acid "Rel homology domain" that contains DNA-binding and dimerization domains. At the C-terminus of the Rel homology domain is a nuclear translocation sequence that is important in the transport of NF-κB from the cytoplasm to the nucleus. In addition, p65 and cRel have potent transactivation domains at their C-termini.

The activity of NF-κB is regulated through its interaction with a class of inhibitors of the IκB family of proteins. This interaction effectively blocks the nuclear localization sequence on the NF-κB protein, thereby preventing migration of the dimer to the nucleus. A wide variety of stimuli activate NF-κB through possibly multiple signaling pathways. These include bacterial products (LPS), certain viruses (HIV-1, HTLV-1), inflammatory cytokines (TNFα, IL-1), and environmental stress. Apparently the same for all stimuli is the phosphorylation and subsequent degradation of IKB. IKB is phosphorylated on its two N-terminal serines by the recently identified IKB kinases (IKK-α and IKK-B). Site-dependent mutagenesis studies suggest that these phosphorylations are important for subsequent NF-κB activation, ie, once phosphorylated proteins are marked for degradation via the ubiquitin-protein kinase pathway. Freed from IκB, active NF-κB complexes can translocate to the nucleus where they selectively associate with preferred gene-specific enhancer sequences. Genes regulated by NF-κB include various cytokines, cell adhesion molecules, and acute phase proteins.

NF-κB is known to play a key role in regulating the expression of numerous proinflammatory mediators, including cytokines, such as IL-6 and IL-8; cell adhesion molecules, such as ICAM and VCAM; and a Nitric Oxide Synthesis Inducing Enzyme (iNOS). These mediators are known to play a role in leukocyte recruitment at sites of inflammation, and in certain inflammatory and autoimmune diseases, iNOS can lead to organ destruction.

The importance of NF-κB in inflammation has been reinforced by studies of airway inflammation, including asthma, where NF-κB has been shown to be activated. This activation may underlie increased cytokine production and leukocyte infiltration in these diseases. In addition, inhaled steroids are known to attenuate airway hyperreactivity and suppress airway inflammatory responses in asthmatics. Given recent findings regarding the inhibitory effects of glucocorticoids on NF-κB, one might expect that these effects are mediated through inhibition of NF-κB.

Additional evidence for the role of NF-κB in inflammatory diseases comes from studies of rheumatoid synovium. Although NF-κB is generally present in the cytosol as an inactive complex, recent immunohistochemical studies have shown that NF-κB is present in the nucleus and is therefore active in cells comprising rheumatoid synovium . Furthermore, NF-κB was shown to be in an activated form in human synoviocytes sensitive to TNF-K. This distribution may underlie the increased production of cytokines and eicosanoids characteristic of this tissue. See Roshak, A.K. et al., J. Biol. Chem., 271, 31496-31501 (1996).

NF-κB/Rel and IκB proteins may also play a role in tumor transformation. Family members are involved in the transformation of cells in vitro or in vivo as a result of overexpression, gene amplification, gene rearrangement or translocation. Additionally, rearrangements and/or amplifications of the genes encoding these proteins are observed in 20-25% of certain human lymphoid neoplasms. Furthermore, the role of NF-κB in regulating apoptosis has been reported to enhance the role of this transcription factor in controlling cell proliferation.

Several NF-κB inhibitors are described in the following documents: C. Wahl et al., J. Clin. Invest. 101(5), 1163-1174 (1998); R.W. Sullivan et al., J.Med.Chem.41, 413-419 ( 1998); J.W. Pierce et al., J. Biol. Chem. 272, 21096-21103 (1997).

The marine natural product hymenialdisine is known to inhibit NF-κB. Roshak, A. et al., JPET, 283, 955-961 (1997). Breton, J.J. and Chabot-Fletcher, M.C., JPET, 282, 459-466 (1997).

Aminoindanones are known compounds. The general preparation of aminoindanone analogues is described in G. Maury, E.-M. Wu. N.H. Cromwell, J. Org. Chem. 1968, 33, 1900-1907. The synthesis of the 3-bromo intermediate is described in B.D.Pearson, R.P.Ayer.N.H.Cromwell, J.Org.Chem.1962,27,3038-3044. The synthesis of 2-benzylideneindanone is described in A.Hassner,N.H.Cromwell,J.Org.Chem.1958,80,893 -900.

We have now discovered a new approach to inhibit the activation of the transcription factor NF-κB using aminoindanones.

Brief description of the invention

It is an object of the present invention to provide a method of treating diseases that can be cured and alleviated by altering the activity of the transcription factor NF-κB.

Accordingly, in one aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the present invention provides a method of treating a disease, wherein the pathology of the disease is therapeutically alleviated by inhibiting NF-KB.

In particular, the present invention provides methods of treating various diseases associated with NF-κB activation, including inflammatory diseases; especially rheumatoid arthritis, inflammatory bowel disease and asthma; skin diseases, including psoriasis and atopic autoimmune diseases; tissue and organ rejection; Alzheimer's disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkin's disease; and certain viral infections, including AIDS; Osteoarthritis; Osteoporosis; and Ataxia Telangiestasia.

Detailed description of the invention

其中：The present invention provides a method for treating diseases related to NF-κB activation, comprising administering the compound of formula I and pharmaceutically acceptable salts, hydrates and solvates thereof to animals requiring such treatment, preferably mammals, most preferably humans:

in:

R ₁ is aryl;

R ₂ is selected from: H, C _1-6 alkyl and aryl;

R ₃ is selected from: C _1-6 alkyl and C _3-8 cycloalkyl; and

R ₂ and R ₃ may together form a heterocyclic ring of 5-7 atoms, the atoms shown are selected from: C, N, O and S.

The present invention preferably provides a method for the treatment of: inflammatory diseases; especially rheumatoid arthritis, inflammatory bowel disease and asthma; skin diseases, including psoriasis and atopic dermatitis; autoimmune diseases; tissue and organ rejection Alzheimer's disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkin's disease; and certain viral infections, including AIDS; osteoarthritis; osteoporosis; and Ataxia Telangiestasia.

Compounds of formula I selected from the following groups are preferably used in the method of the invention:

3-[(N-butyl)amino]-2-benzylideneindanone;

3-[(N-cyclohexyl)amino]-2-benzylidene indanone;

3-morpholinyl-2-benzylidene indanone; and

3-Piperidinyl-2-benzylidene indanone.

definition

The present invention includes the use of all hydrates, solvates, complexes and prodrugs of the compounds of the present invention. A prodrug is any covalently bonded compound that releases the active parent drug of formula I in vivo. If one chiral center or another form of isomeric center is present in a compound of the invention, the invention includes all forms of such isomers, including enantiomers and diastereomers. Compounds of the invention which contain a chiral center may be used as racemic mixtures enriched in one enantiomer or racemic mixtures which may be separated using well known techniques, or the enantiomers may be used individually. In cases where compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are included within the scope of the invention. In cases where a compound can exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form whether present in equilibrium or predominantly in one form should be included in Book

within the scope of the invention.

The meanings of any substituents present in formula I or sub-formulae thereof are independent of each other and, unless otherwise stated, in any other case, have the meaning of any other substituent.

"C _1-6 alkyl" herein refers to substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl, pentyl, n-pentyl , isopentyl, neopentyl and hexyl and their simple aliphatic isomers. Any C _1-6 alkyl can optionally be independently replaced by one or two halogens, SR', OR', N(R') ₂ , C(O)N(R') ₂ , carbamoyl or C _{1- 4} alkyl substitutions, wherein R' is C _1-6 alkyl.

"C _3-8 cycloalkyl" herein refers to substituted and unsubstituted cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, cycloheptyl and cyclooctyl and their simple aliphatic isomers body. Any C _3-8 cycloalkyl group can optionally be independently replaced by one or two halogens, SR', OR', N(R') ₂ , C(O)N(R') ₂ , carbamoyl or C _{1 -4} alkyl substitution, wherein R' is C _1-6 alkyl.

"Halogen" herein includes F, Cl, Br and I.

"Ar" or "aryl" herein includes phenyl or naphthyl, which may be optionally substituted independently by one or more of the following groups: Ph-C _0-6 alkyl, Het-C _0-6 Alkyl, C _1-6 alkyl, C _1-6 alkoxy, Ph-C _0-6 alkoxy, Het-C _0-6 alkoxy, OH, CN, CO ₂ R' or halogen. C ₀ Alkyl means that no alkyl group is present in the moiety. Therefore, Ar-C ₀ alkyl is equivalent to Ar. Two C _1-6 alkyl groups can combine to form a 5-7 membered ring, which can be saturated or unsaturated and fused to the Ar ring. Ph can be optionally substituted with one or more C _1-6 alkyl, C _1-6 alkoxy, OH, CO ₂ R' or halogen.

"Het" or "heterocycle" as used herein means a stable 5-7 membered monocyclic ring, which may be saturated or unsaturated, consisting of carbon atoms and one to three heteroatoms selected from N, O and S and wherein the nitrogen and sulfur heteroatoms are optionally oxidized, and the nitrogen heteroatoms are optionally quaternized; and include any bicyclic group in which any heterocycle as defined above is fused to a benzene ring. The heterocycle may be attached at any heteroatom or carbon atom, resulting in a stable structure, and may be optionally substituted with one or more groups selected from: Ph-C _0-6 alkane group, Het-C _0-6 alkyl, C _1-6 alkyl, C _1-6 alkoxy, Ph-C _0-6 alkoxy, Het-C _0-6 alkoxy, OH or CN. Two C _1-6 alkyl groups may combine to form a 5-7 membered ring, which may be saturated or unsaturated and fused to the Het ring. Ph can be optionally substituted with one or more C _1-6 alkyl, C _1-6 alkoxy, OH, CO ₂ R' or halogen. Examples of such heterocyclic rings include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepanyl Gene group, pyrrolyl, 4-pyridonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, iso Oxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinidinyl, indolyl, quinolinyl, isoquinolyl, benzimidazolyl, benzopyranyl, benzo Oxazolyl, furyl, pyranyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzoxazolyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone and oxadiazolyl rings.

Preparation

The compounds used in the methods of the present invention can be conveniently prepared by the methods shown in Scheme 1 below.

The general preparation of aminoindanone analogues is described in G. Maury, E.-M. Wu. N.H. Cromwell, J. Org. Chem. 1968, 33, 1900-1907. The synthesis of the 3-bromo intermediate is described in B.D.Pearson, R.P.Ayer.N.H.Cromwell, J.Org.Chem.1962, 27, 3038-3044. The synthesis of 2-benzylideneindanone is described in A.Hassner, N.H.Cromwell, J.Org.Chem 1958, 80, 893-900. General preparation method:

The general preparation is shown in Scheme 1. Treatment of indanone with benzaldehyde in base gives 2-benzylidene indanone. Bromination of 2-benzylidene indanone with NBS in _CCl4 gave 3-bromo-2-benzylidene indanone. Treatment of 3-bromo-2-benzylidene indanones with alkyl primary or secondary amines in benzene affords 3-alkylamino-2-benzylidene indanones.

Reaction Scheme 1

With respect to the process for the preparation of compounds of formula I shown in Scheme 1 above, it will be clear to those of ordinary skill in the art that all novel intermediates required for the preparation of compounds of formula I are intended to be included in the present invention.

The starting materials used herein are commercially available or can be prepared by conventional methods well known to those of ordinary skill in the art, see general references, eg COMPENDIUM OF ORGANICS SYNTHETIC METHODS, Vol. I-VI (published by Wiley-Interscience).

The acid addition salts of the compounds of formula I can be prepared in a standard manner from the parent compound and an excess of an acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, acetic acid, tris Fluoroacetic acid, maleic acid, succinic acid or methanesulfonic acid. It is also acceptable for certain compounds to form internal salts or zwitterions. Cationic salts are prepared by treating the parent compound with an excess of a basic reagent containing the appropriate cation, such as a hydroxide, carbonate, or alcoholate; or with a suitable organic amine. Examples of cations present in pharmaceutically acceptable salts are eg Li ⁺ , Na ⁺ , K ⁺ , Ca ⁺⁺ , Mg ⁺⁺ and NH ₄ ⁺ . Examples of anions present in pharmaceutically acceptable salts are halides, sulfate, phosphate, alkanoates (eg acetate and trifluoroacetate), benzoate and sulfonates (eg methanesulfonate).

The present invention provides a pharmaceutical composition, which contains the compound of formula I and a pharmaceutically acceptable carrier, diluent or excipient. Therefore, the compounds of formula I are useful in the preparation of medicaments. Pharmaceutical compositions of compounds of formula I prepared as described above may be formulated as solutions or lyophilized powders for parenteral administration. Powders can be reconstituted by adding suitable diluents or other pharmaceutically acceptable carriers just before use. Liquid preparations can be buffered isotonic aqueous solutions. Examples of suitable diluents are physiological isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulations are especially suitable for parenteral administration, but may also be used orally or in metered dose inhalers or nebulisers for insufflation. It may also be advisable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

Alternatively, the compounds may be formulated for oral administration in capsules, tablets or prepared in the form of emulsions or syrups. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or increase the stability of the composition or to aid in the preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, gypsum, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier also includes a sustained release material such as glyceryl monostearate or glyceryl distearate, or a mixture thereof with a wax. The amount of solid carrier used may vary but preferably will contain from about 20 mg to about 1 g of solid carrier per dosage unit. Pharmaceutical preparations are prepared by conventional pharmaceutical techniques, preparing tablets, including grinding, mixing, granulating and compressing, if necessary; preparing hard gelatin capsules, including grinding, mixing and filling. When a liquid carrier is used, the formulation can be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. Such liquid preparations can be administered directly orally (p.o.) or filled into soft gelatin capsules.

For rectal administration, the compounds of the invention can also be mixed with excipients such as coconut oil, glycerin, gelatin or polyethylene glycols and molded into suppositories.

The methods of this invention involve topical application of a compound of formula I. Topical administration refers to non-systemic administration and includes topical application to the epidermis, buccal buccal administration of the compounds of the invention and instillation of such compounds into the ears, eyes and nose, wherein the dose of the compound does not significantly enter the bloodstream. Systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration. The amount of a compound of formula I (hereinafter referred to as active compound) required to produce a therapeutic or prophylactic effect by topical administration varies with the compound selected, the nature and severity of the indication to be treated and the animal being treated, and of course ultimately determined by Physician decides.

Although the active ingredient can be administered as a raw chemical, it is preferably administered as a pharmaceutical formulation. For topical administration, the active ingredient may constitute 0.01%-5.0% of the formulation.

Whether for veterinary or human use, the topical formulations of the present invention comprise the active ingredient together with one or more acceptable carriers, and optionally other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient.

Formulations suitable for topical administration include liquid or semisolid formulations suitable for penetration through the skin into the desired area to be treated, such as liniments, lotions, creams, ointments or pastes, and for ophthalmic, aural or nasal administration. drops.

The drops of the present invention may contain sterile aqueous or oily solutions or suspensions, which may be prepared by dissolving the active ingredient in an aqueous solution of a suitable bactericidal and/or fungicidal and/or any other suitable preservative, which The aqueous solution preferably contains a surfactant. The resulting solution is then clarified by filtration, transferred to a suitable container, which is then sealed and sterilized by autoclave or at 90-100°C for 1.5 hours. Alternatively, sterilization may be performed by filtration followed by transfer to containers using aseptic technique. Examples of bactericides or fungicides suitable for inclusion in the drops include phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for preparing oily solutions include glycerol, dilute ethanol and propylene glycol.

Lotions of the present invention include those suitable for skin or eye application. Eyewashes may comprise sterile aqueous solutions, optionally containing an antiseptic, which may be prepared in a manner analogous to the preparation of drops. Lotions or liniments applied to the skin may also include substances that accelerate drying and cool the skin, such as alcohol or acetone; and/or humectants, such as glycerin or oils such as castor oil and peanut oil.

The creams, ointments or pastes of the present invention are semi-solid formulations of the active ingredient for external application. Semi-solid formulations may be prepared by mixing the active ingredient in finely divided form with an oily or non-oily base, either in finely divided form itself or in solution or suspension in aqueous or non-aqueous liquids, with the aid of suitable machines. Bases may include hydrocarbons, such as hard, soft, or liquid paraffin, glycerin, beeswax, metallic soaps; slime; oils of natural origin, such as almond, corn, peanut, castor, or olive oil; lanolin or its derivatives , or mixtures of fatty acids such as stearic acid or oleic acid and alcohols such as propylene glycol or polyethylene glycol. The formulation may incorporate any suitable surfactant, such as anionic, cationic or nonionic surfactants, such as sorbitan esters or polyethylene oxide derivatives thereof. Suspending agents, such as natural gums, cellulose derivatives, or inorganic materials, such as siliceous silicas, and other ingredients such as lanolin, may also be present.

Practicality of the invention

Compounds of formula I are useful inhibitors of NF-κB. The present invention provides useful compositions and formulations of the compounds, including pharmaceutical compositions and formulations of the compounds.

The present invention also provides a method for treating diseases associated with NF-κB activation, the method comprising administering a compound of formula I to an animal, especially a mammal, most preferably a human, in need of such treatment. The present invention preferably provides methods of treatment involving inflammatory diseases; especially rheumatoid arthritis, inflammatory bowel disease and asthma; skin diseases, including psoriasis and atopic dermatitis; autoimmune diseases; tissue and organ rejection Alzheimer's disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkin's disease; and certain viral infections, including AIDS; osteoarthritis; osteoporosis; and Ataxia Telangiestaisa.

For acute treatment, the compounds of formula I are preferably administered parenterally. The intravenous infusion of the compound of the present invention in 5% dextrose aqueous solution or 5% dextrose physiological saline solution is the most effective, of course intramuscular bolus injection is also effective. The dosage for parenteral administration is typically about 0.01-50 mg/kg; preferably 0.1-20 mg/kg, to maintain the plasma concentration of the drug at a concentration effective to inhibit NF-κB activation. Compounds may be administered one to four times daily to obtain a total daily dosage of about 0.4-80 mg/kg/day. The precise dosage and optimal route of administration of a therapeutically effective compound of the invention are readily determined by one of ordinary skill in the art by comparing blood levels of the compound with the concentration required to produce a therapeutic effect.

Compounds of Formula I may also be administered orally to a patient at concentrations sufficient to inhibit NF-κB or treat any of the other indications disclosed herein. Pharmaceutical compositions containing the compounds are generally administered at an oral dose of about 0.1-50 mg/kg (in keeping with the indications for the patient). Oral dosages are preferably about 0.5-20 mg/kg.

Compounds of formula I may also be administered topically to a patient at concentrations sufficient to inhibit NF-KB or treat any of the other indications disclosed herein. Pharmaceutical compositions containing the compounds are generally administered as topical formulations at about 0.01%-5% w/w.

No unacceptable toxic side effects are expected when the compounds of the invention are administered in accordance with the invention.

The ability of the compounds described herein to inhibit NF-κB activation is clearly demonstrated by their ability to inhibit reporter genes that drive NF-κB (see Table 1). The practicability of the NF-κB inhibitors of the present invention in the treatment of diseases is predicated on the importance of NF-κB activation in various diseases.

Table 1 Inhibition of NF-κB-driven reporter gene activity

NF-κB plays a key role in regulating the expression of numerous inflammatory mediators, including cytokines such as IL-6 and IL-8 (Mukaida et al., 1990; Liberman and Baltimore, 1990; Matsusaka et al., 1993) ; cell adhesion molecules such as ICAM and VCAM (Marui et al., 1993; Kawai et al., 1995; Ledebur and Parks, 1995); and nitric oxide synthesis inducing enzyme (iNOS) (Xie et al., 1994; Adcock et al., 1994). (All these documents are attached at the end of this section). These mediators are known to play a role in leukocyte recruitment at sites of inflammation, and iNOS can lead to organ destruction in certain inflammatory and autoimmune diseases (McCartney-Francis et al., 1993; Kleemann et al., 1993). Importantly, the compounds described herein inhibit IL-8 synthesis and production of nitric oxide, a product of iNOS activity (see Table 2).

Anti-inflammatory activity of compound 2 in Table 2 Table 1 in vitro IL-1-induced PGE2 production in RSF IC ₅₀ =1μM in vivo Inflammatory cell infiltration of ear swelling in phorbol ester-induced ear inflammation 85% inhibition @1mg/ear 29% inhibition @1mg/ear

Evidence for an important role of NF-κB in inflammatory diseases has been obtained from studies of patients with asthma. Bronchial biopsies taken from mild allergic asthma compared with those taken from normal non-allergic controls showed submucosa staining for activated NF-κB, total NF-κB, and NF-κB-regulated cytokines such as GM-CSF and TNF-α significantly increased the number of cells (Wilson et al., 1998). In addition, the percentage of immunoreactivity expressing NF-KB vectors was elevated as was IL-8 immunoreactivity in the epidermis of biopsy samples (Wilson et al., 1998). Thus, IL-8 production was inhibited through inhibition of NF-κB, as evidenced by the expected beneficial effects of these compounds on airway inflammation.

Recent studies have shown that NF-κB also plays a key role in the pathogenesis of inflammatory bowel disease (BID). Activated NF-κB has been observed in colonic biopsy samples taken from patients with Crohn's disease and ulcerative colitis (Ardite et al., 1998; Rogler et al., 1998; Schreiber et al., 1998). Activation was evident in inflamed mucosa; it was not evident in non-inflamed mucosa (Ardite et al., 1998; Rogler et al., 1998) and was associated with increased expression of IL-8 mRNA at these same sites (Ardite et al., 1998). Furthermore, corticosteroid treatment strongly inhibited intestinal NF-KB activation and attenuated colonic inflammation (Ardite et al., 1998; Rogler et al., 1998; Schreiber et al., 1998). Additionally, IL-8 production was inhibited by inhibition of NF-κB, as evidenced by the expected beneficial effects of these compounds in inflammatory bowel disease.

Animal models of gastrointestinal inflammation further support the notion that NF-κB is a key regulator of colonic inflammation. NF-κB activity was observed in the lamina propria of macrophages induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice bearing p65, a major component of the activation complex (Neurath et al., 1996; Neurath and Pettersson, 1997). Local administration of p65 antisense abolished the signs of established colitis in treated animals without signs of toxicity (Neurath et al., 1996; Neurath and Pettersson, 1997). Therefore, small molecule NF-κB inhibitors can be predicted to be useful in the treatment of IBD.

Further evidence for the role of NF-κB in inflammatory diseases comes from studies of rheumatoid synovium. Although NF-κB is normally present as an inactive cytoplasmic complex, recent immunohistochemical studies have shown that NF-κB is also present in the nucleus and thus active in cells including human rheumatic synovium (Handel et al. , 1995; Marok et al., 1996; Sioud et al., 1998), in addition it is present in the nucleus in animal models of the disease and is thus active (Tsao et al., 1997). Staining is associated with type A synoviocytes and vascular endothelium (Marok et al., 1996). Additionally, in cultured synoviocytes (Roshak et al., 1996; Miyazawa et al., 1998) and in cultures of synoviocytes stimulated with IL-1β or TNF-α (Roshak et al., 1996; Fujkawa et al., 1996; Roshak et al., 1997), constitutive activation of NF-κB was observed. Thus, NF-κB activation may underlie the increased cytokine production and leukocyte infiltration characteristic of inflammatory synovium. It could be predicted that the ability of these compounds to inhibit NF-KB and thus eicosanoid production by these cells would have a beneficial effect on rheumatoid arthritis (see Table 2).

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Neurath MF, Pettersson S, Meyer zum Buschenfelde K-H, Strober W (1996) Topical administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-κB abolishes established experimental colitis in mice, Nature Med 2:998-1004.

Neurath MF, Pettersson S (1997) The dominant role of NF-κB p65 in the pathogenesis of chronic enteritis. Immunolbiol 198:91-98. Handel ME, McMorrow LB, Gravallese EM (1995) Nuclear factor-κB in rheumatoid synovium; localization of p50 and p65, Arthritis Rheumatism 38:1762-1770.

Marok R, Winyard PG, Coumbe A, Kus ML, Gaffney K, Blades S, Mapp PI, Morris CJ, Blake DR, Kaltschmidt C, Baeuerle PA (1996) Activation of the transcription factor nuclear factor-κB in human inflamed synovial tissue, Arthritis Rheumatism 39:583-591.

Sioud M, Mellbye O, Forre O (1998) Analysis of NF-κB p65 subunit, Fas antigen, Fas ligand, and Bcl-2-related proteins in RA synovium and polyarticular JRA. ClinExpRheumatol 16:125-134.

Tsao PW, Suzuki T, Totsuka R, Murata T, Takagi T, Ohmachi Y, Fjimura H, Takata I (1997) Effect of dexamethasone on expression of activated NF-κB in adjuvant arthritis, Clin Immunol Immunopathol 83: 173-178.

Roshak AK, Jackson JR, McGough K, Chabot-Fletcher M, Mochan E, Marchall L (1996) Treatment of different NF-κB proteins alters interleukin-1β-induced prostaglandin E2 formation in human rheumatic synovial fibroblasts , J Biol Chem 271:31496-31501.

Miyazawa K, Mori A, Yamamoto K, Okudaira H (1998) Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes; spontaneous activation of NF-κB and CBF1, Am J Pathol 152, 793-803

Fujisawa K, Aono H, Sasunuma T, Yamamoto K, Mita S, Nishiola K (1996) Activation of the transcription factor NF-κB in human synoviocytes in response to tumor necrosis factor alpha, Aathritis Rheumatism 39:197-203.

Roshak AK, Jackson JR, Chabot-Fletcher M, Marshall L (1997) NF-κB-mediated inhibition of interleukin-1β-stimulated prostaglandin E2 formation by the marine natural product hymenialdisine, J Pharmacol Exp Therapeut 283:955-961.

biological analysis

Compounds of the invention are tested in one of several biological assays to determine the concentration of compound required to produce a given pharmacological effect.

NF-κΒ activity was analyzed using a cell-based luciferase reporter assay as described in Breton, JJ and Chabot-Fletcher, MC, JPET, 282, 459-466 (1997). Briefly, U937 human histiocytic lymphoid cells permanently transfected with an NF-κB reporter plasmid (see below) were cultured in the above medium supplemented with 250 μg/ml geneticin (G418 sulfate, Life Technologies, Grand Island, NY). cell line. Luciferase reporter assays were performed in transfected U937 clone cells. After centrifugation at 300xg twice (5 minutes each), resuspend in RPMI 1640 containing 10% FBS to 1 x ¹⁰⁶ cells/ml. 1 mL aliquots of this suspension were added to wells of a 24-well plate. Compound or dimethyl sulfoxide (DMSO) vehicle (1 μl) was added to the appropriate wells and the plate was incubated at 37° C., 5% CO ₂ for 30 minutes. Stimulators (5 ng/ml TNFα, 100 ng/ml LPS or 0.1 μM PMA) were added, the samples were incubated at 37 °C, 5% CO for 5 min, transferred to 1.9 ml polypropylene _tubes , and centrifuged at 200 x g for 5 min. minute. Cell pellets were washed twice with 1 ml PBS without Ca2 ⁺ and Mg2 ⁺ and centrifuged as indicated above. The resulting cell pellet was lysed in 50 μl of 1× Lysis Buffer (Promega Corporation, Madison, WI), vortexed, and incubated at room temperature for 15 minutes. 20 μl aliquots of lysates were transferred to opaque white-bottom 96-well culture plates (Wallac Inc., Gaithersburg, MD) and analyzed for luciferase in a Micro Lumant LB 96 P luminometer (EG&G Berthold, Bad Wilbad, Germany) generation. The luminometer dispenses 100 μl of Luciferase Assay Reagent (Promega Corporation, Madison, WI) into each well and records the overall light emission for 20 seconds. The amount of light emitted is measured in relative light units (RLUs).

The presence of NF-κB protein in the nucleus can also be estimated by measuring NF-κB activity using electrophoretic mobility shift assay (EMSA). The analyzed cells were cultured at 1×10 ⁶ /ml. Cells were captured by centrifugation, washed with PBS without Ca ²⁺ and Mg ²⁺ , and suspended in PBS with Ca ²⁺ and Mg ²⁺ at a density of 1×10 ⁷ cells/ml. To determine the effect of compounds on NF-κB activation, cell suspensions were treated with different concentrations of drug or vehicle (DMSO, 0.1%) for 30 minutes at 37°C, and then stimulated with TNFα (5.0 ng/ml) for 15 minutes. Extracts of cells and nuclei were prepared as follows. In summary, at the end of treatment, cells (1 x ¹⁰⁷ cells) were washed twice with PBS without Ca2 ⁺ and Mg2 ⁺ . The resulting cell pellet was resuspended in 20 μl buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl ₂ , 0.5 mM dithiothreitol (DTT) and 0.1% NP-40) and kept on ice Keep warm for 10 minutes. Nuclei were pelleted by microcentrifugation at 3500 rpm for 10 minutes at 4°C. The supernatant was collected as a cell extract, and the nuclear pellet was resuspended in 15 μl buffer C (20 mM Hepes (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl ₂ , 25% glycerol, 0.2 mM EDTA, 0.5MM DTT and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). After gently stirring the suspension at 4°C for 20 minutes, it was microcentrifuged at 14000 rpm for 10 minutes at 4°C. The supernatant was collected and diluted to 60 μl with buffer D (20 mM Hepes (pH 7.9), 50 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5MM DTT and 0.5 mM PMSF). All samples were stored at -80°C until analysis. According to the method of Bradford (Bradford, 1976), the protein concentration of the extract was determined with BioRad reagent.

Nuclear extracts of treated cells as described above were used to assess the effect of compounds on transcription factor activation using electrophoretic mobility shift assay (EMSA). The double-stranded NF-κB consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was labeled with _T4 polynucleotide kinase and [g- ^32P ]ATP. The combined mixture (25 μl) contains 10 mM Hepes-NaOH (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.3 mg/ml fetal bovine serum Albumin and 1 μg poly(dI-dC).poly(dI-dC). The bound mixture (10 μg nuclear extracted protein) was incubated with 0.5 ng ³² P-labeled oligonucleotides (50,000-100,000 cpm) in the presence or absence of unlabeled competitor for 20 minutes at room temperature, after which This mixture was loaded onto a 4% polyacrylamide gel prepared in 1X Tris borate/EDTA and electrophoresed at 200 volts for 2 hours. After electrophoresis, the gel is dried and contacted with a membrane to measure the binding reaction.

The effect of compounds on IKB phosphorylation can be monitored by Western blot. Cell extracts were subjected to sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels (BioRad, Hercules, CA) and proteins were transferred to nitrocellulose membranes (Hybond ^1m -ECL, Amersham Corp., Arlington Heights, IL). Immunoblot analysis was performed with polyclonal rabbit antibodies directed against IκBα or IκBβ, followed by a peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham Corp., Arlington Heights, IL). Immunoreactive bands were monitored with an enhanced chemiluminescence (ECL) analysis system (Amersham Corp., Arlington Heights, IL).

Primary cultures of human RSF were used to evaluate the effect of human synovial fibroblasts (RSF) on eicosanoid production. RSF was obtained by enzymatic digestion of synovium taken from adult human rheumatic patients. Cells were cultured at 37°C and 5% CO in Earl's Minimal Essential Medium ( _EMEM ) which also contained 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml chain Mycin (GIBCO, Grand Island, NY). To obtain a more homogeneous population of type I fibroblasts, use cultures of cells at passages 4-9, and for some studies, plate fibroblasts at a density of 5 x ¹⁰⁴ cells/ml into 24 wells with a diameter of 16 mm Culture plates (Costar, Cambridge, MA). Cells were exposed to optimal doses of IL-Ιβ (1 ng/ml; Roshak et al., 1996a) (Genzyme, Cambridge, MA) for the indicated times. Drugs (1%) in DMSO vehicle were added to the cell cultures 15 minutes before the addition of IL-1. The level of prostaglandin E2 pooled in the cell-free medium at the end of the culture was measured directly with an enzyme immunoassay (EIA) kit purchased from Cayman Chemical Co. (Ann Arbor, MI). Prepare samples and standard dilutions in the experimental medium.

In vivo anti-inflammatory activity was evaluated using a phorbol ester-induced mouse ear inflammation model. Phorbol cinnamate acetate (PMA) (4 μg/20 μl acetone) was applied to the inner and outer surfaces of the left ear of male Balb/c mice (6/group) (Charles River Breeding Laboratories, Wilmington, MA) for 4 h Afterwards, compounds dissolved in 25 μl of acetone were applied on the same ear. After 20 hours, the thickness of both ears was measured with a graduated micrometer (Mitutoyo, Japan), and a second dose of compound was applied topically. After 24 hours, the thickness of the ear was measured, and the data was expressed as the thickness change (×10 ^-3 cm) between the treated ear and the untreated ear. The inflamed left ear was then excised and stored at -70°C until analysis for analysis of myeloperoxidase (MPO) activity, a measure of inflammatory cell infiltration.

Infiltration of inflammatory cells was assessed by measuring myeloperoxidase activity present in inflamed ear tissue. Partially thawed ear tissue was minced and then homogenized (10% w/v) in 50 mM phosphate buffer (pH 6) containing 0.5% HTAB using a Tissumizer homogenizer (Tekmar Co., Cincinnati, OH). Homogenates were subjected to three freeze-thaw cycles followed by brief sonication (10 sec). MPO activity in the homogenate was determined as follows. The appearance of the colored product of the MPO-dependent reaction of o-dimethoxyaniline (0.167 mg/ml, Sigma Chemical, St. Louis, MO) and hydrogen peroxide (0.0005%) was measured spectrophotometrically at 460 nm. The MPO activity of the supernatant was quantified kinetically (sampling at 15 second intervals and measuring the change in absorbance for 3 minutes) using a Bechman DU-7 spectrophotometer and a kinetic analysis device (Beckman Instruments, Inc., Sommerset, NJ). One unit of MPO activity is defined as the decomposition of one micromole of peroxide per minute at 25°C.

The effect on inflammation-mediated cartilage breakdown was determined in an in vitro cartilage dissociation block system. In this model, bovine articular cartilage fragments or the fragments were incubated with rHuIL-la in the presence or absence of test compounds for 4 days/96 hours to promote cartilage breakdown. The supernatant was removed for nitric oxide analysis. Nitric oxide was determined by Greiss reaction and reading the data at 530nm with a spectrophotometer. This reaction measures nitrogen dioxide ( _NO2 ), which is a stable end product of nitric oxide.

Generalize

NMR spectra were measured using a Bruker AM 250, Bruker 300 or Bruker AC 400 spectrometer at 250, 300 or 400 Mhz, respectively. CDCl ₃ is deuteriochloroform, DMSO-d ₆ is hexadeuteriodimethylsulfoxide, CD ₃ OD is tetradeuteriomethanol. Chemical shift values are reported in parts per million downfield compared to the internal standard tetramethylsilane. Abbreviations in NMR data are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, app = clearly visible peak, br=broad peak. J denotes the NMR coupling constant measured in Hertz. Continuous wave infrared spectroscopy (IR) was recorded on a Perkin Elmer683 infrared spectrometer, and Fourier transform infrared spectroscopy (FTIR) was recorded on a Nicolet Impact 400D infrared spectrometer. IR and FTIR were recorded in transmission mode, and band positions were recorded in units of inverse wavenumber (cm ^-1 ). Mass spectra were determined using fast atom bombardment (FAB) or electrospray (ES) ionization techniques on a VG 70 FE, PESyx API III or VG ZAB HF instrument. Elemental analysis was performed with a Perkin-Elmer 240C elemental analyzer. Melting points were determined on a Thomas-Hoover melting point apparatus without correction. All temperatures are reported in degrees Celsius.

Thin-layer chromatography was performed with Analtech Silica Gel GF and E. Merck Silica Gel 60F-254 thin-layer plates. Flash chromatography and gravity chromatography were performed with E. Merck Kieselgel 60 (230-400 mesh) silica gel.

As indicated below, certain starting materials were purchased from Aldrich Chemical Co., Milwaukee, Wisconsin.

Example

In the following synthesis examples, temperatures are expressed in degrees Celsius (°C). All raw materials were obtained from commercial sources unless otherwise stated. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. These examples illustrate the invention without limiting its scope. What the inventor seeks protection is the claims mentioned below.

Example 1

Preparation of 3-[(N-butyl)amino]-2-benzylideneindanone

a) 2-benzylidene indanone

According to the method of A.Hassner, NH Cromwell, J.Org.Chem.1958,80,893-900, benzaldehyde (6.05ml, 53.6mmol) was added to dihydroindanone (Aldrich, 7.08g, 53.6mmol) at 0°C ) in ethanol and KOH (600 mg, 10.6 mmol), the reaction was placed in the refrigerator overnight. The reaction mixture was filtered, washed with 50% aqueous ethanol, and then recrystallized in hot ethanol to obtain 5.84 g of 2-benzylidene indanone. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.93 (d, 1H, J=7.7), 7.75-7.35 (m, 8H), 4.08 (s, 2H).

b) 3-bromo-2-benzylindanone

According to the method of BDPearson, RPAyer, NH Cromwell, J.Org.Chem.1962,27,3038-3044, in carbon tetrachloride (15ml), the compound (1.0g, 4.55mmol) that embodiment 1 (a) obtains Treat with NBS (810 mg, 4.55 mmol) and benzoyl peroxide (50 mg) and heat the mixture under reflux with a heat lamp for 1 hour. The reaction was cooled, filtered and the filtrate evaporated to give 3-bromo-2-benzylideneindanone which was used without purification. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.1-7.2 (m, 10H), 6.40 (s, 2H).

c) 3-[(N-butyl)amino]-2-benzylidene indanone

According to the method of G.Maury, E.-M.Wu.N.H.Cromwell, J.Org.Chem.1968,33,1900-1907, the compound obtained in embodiment 1(b) was treated with n-butylamine ( 300 μl, 3.04 mmol) and the solution was stirred at room temperature for 24 hours. The reaction was evaporated and the residue was purified by flash chromatography (silica gel, 10% ethyl acetate in hexanes) to give 353 mg of 3-[(N-butyl)amino]-2-benzylideneindanone. Treatment of a portion of the free base with 1N hydrochloric acid in ether in ether gave the hydrochloride salt as a solid. ES-MS(M+H)+m/e 292; H NMR(300MHz, CDCl)δ8.2(d,1H,J=7.0Hz),9.03(s,1H),7.98(d,1H,J= 6Hz),7.84(t,1H,J=6Hz),7.75-7.50(m,7H),6.7(brs,1H),2.48(brs,1H),2.28(brs,1H),1.6-1.3(m, 2H), 0.95-0.8(m, 2H), 0.65(t, 3H, J=6.5Hz).

The above specification and examples fully disclose how to make and use the compounds of the invention. However, the invention is not limited to the specific embodiments described above, but includes all modifications thereof within the scope of the invention. Various journals, patents, and other publications cited herein contain descriptions of the prior art and are hereby incorporated by reference in their entirety.

Claims (18)

1. A pharmaceutical composition containing a compound of formula I

in: R ₁ is aryl; R ₂ is selected from: H, C _1-6 alkyl and aryl; R ₃ is selected from: C _1-6 alkyl and C _3-8 cycloalkyl; and R ₂ and R ₃ can together form a heterocyclic ring of 5-7 atoms, the atoms shown are selected from: C, N, O and S; And a pharmaceutically acceptable carrier, diluent or excipient. 2. The pharmaceutical composition of claim 1, wherein _R2 is H. 3. The pharmaceutical composition of claim 2, wherein _R3 is selected from the group consisting of butyl and cyclohexyl. 4. The pharmaceutical composition of claim 1, wherein R ₂ and R ₃ can combine together to form a heterocyclic ring with 5-7 atoms, and the atoms are selected from C, N, O and S. 5. The pharmaceutical composition of claim 4, wherein _R3 is selected from morpholinyl and piperidinyl. 6. The pharmaceutical composition of claim 1, wherein said compound is selected from the group consisting of: 3-[(N-butyl)amino]-2-benzylideneindanone; 3-[(N-cyclohexyl)amino]-2-benzylidene indanone; 3-morpholinyl-2-benzylidene indanone; and 3-Piperidinyl-2-benzylidene indanone. 7. A method of inhibiting NF-κB comprising administering an effective amount of a compound of formula I to a patient in need of such inhibition: wherein:

R ₁ is aryl; R ₂ is selected from: H, C _1-6 alkyl and aryl; R ₃ is selected from: C _1-6 alkyl and C _3-8 cycloalkyl; and R ₂ and R ₃ may together form a heterocyclic ring of 5-7 atoms, the atoms shown are selected from: C, N, O and S. 8. The method of claim 7, wherein said compound is selected from the group consisting of: 3-[(N-butyl)amino]-2-benzylideneindanone; 3-[(N-cyclohexyl)amino]-2-benzylidene indanone; 3-morpholinyl-2-benzylidene indanone; and 3-Piperidinyl-2-benzylidene indanone. 9. A method for treating diseases characterized by overactivation of NF-κB, comprising inhibiting said overactivation by administering an effective amount of a compound of formula I and pharmaceutically acceptable salts, hydrates and solvates thereof to a patient in need of such treatment in: R ₁ is aryl; R ₂ is selected from: H, C _1-6 alkyl and aryl; R ₃ is selected from: C _1-6 alkyl and C _3-8 cycloalkyl; and R ₂ and R ₃ may together form a heterocyclic ring of 5-7 atoms, the atoms shown are selected from: C, N, O and S. 10. The method of claim 9, wherein said compound is selected from the group consisting of: 3-[(N-butyl)amino]-2-benzylideneindanone; 3-[(N-cyclohexyl)amino]-2-benzylidene indanone; 3-morpholinyl-2-benzylidene indanone; and 3-Piperidinyl-2-benzylidene indanone. 11. The method of claims 9 and 10, wherein said disease is an inflammatory disease. 12. The method of claim 14, wherein the disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease and asthma. 13. The method of claims 9 and 10, wherein said disease is a skin disease. 14. The method of claim 13, wherein said disease is selected from psoriasis and atopic dermatitis. 15. The method of claims 9 and 10, wherein said disease is selected from the group consisting of: autoimmune disease; tissue and organ rejection; Alzheimer's disease; stroke; atherosclerosis; restenosis; osteoarthritis; pectinosis; and Ataxia Telangiestaisa. 16. The method of claims 9 and 10, wherein said disease is cancer. 17. The method of claim 16, wherein said cancer is Hodgkin's disease. 18. The method of claims 9 and 10, wherein said disease is AIDS.